1. Field of the Invention
The teachings herein are generally directed to a method of enhancing the genetic stability of parvovirus vectors.
2. Description of Related Art
Parvoviruses are among the smallest, simplest eukaryotic viruses and fall into two groups: defective viruses that are dependent on a helper virus for replication; and autonomous, replication-competent viruses. Adeno-associated viruses are non-pathogenic, helper-dependent members of the parvovirus family. One of the identifying characteristics of this group of viruses is the encapsidation of a single-stranded DNA (ssDNA) genome. In the case of AAV, the separate plus or minus polarity strands are packaged with equal frequency, and either is infectious. At each end of the ssDNA genome, a palindromic terminal repeat (ITR) structure base-pairs upon itself into a hairpin configuration. This serves as a primer for cellular DNA polymerase to synthesize the complementary strand after uncoating in the host cell. Being helper dependent, the adeno-associated viruses generally require a helper virus for a productive infection.
Recombinant adeno-associated virus (rAAV) vectors have great potential for use in nucleic acid delivery applications, since rAAV vectors provide a number of advantages over other viral vectors. For example, rAAV vectors are capable of transducing nondividing cells and do not induce an immune response that eliminates the host cells. Like the wild type virus, nucleic acid delivery vectors derived from adeno-associated virus (AAV) can package and deliver single-stranded DNA genomes. Unfortunately, although otherwise very promising, the conventional AAV vectors have major limiting factors.
The main problem with the current generation of AAV gene transfer/therapy vectors is their genetic instability. Conventional AAV vectors typically consist of a recombinant gene expression cassette (promoter—gene—termination signal), flanked by the inverted terminal repeats (ITRs) of AAV. The ITRs are typically 145 bp long sequences derived from the very ends of the AAV genome and serve as DNA replication as well as packaging (or encapsidation) signals. In the current generation of AAV vectors, these ITRs are usually derived from a particular AAV sero/genotype, AAV-2 (the prototype of the AAV family) and are identical to each other. Such vectors can accommodate up to ˜5.2 kb of foreign DNA and are usually referred to as single-stranded AAV (ssAAV, wildtype AAV is also a single-stranded virus).
Recent reports show that this inherent limitation of AAV vectors can be overcome by the use of genomes that are half the length of the virus wild-type, and these genomes can be packaged as dimeric DNA molecules in an inverted repeat configuration (See Hirata and Russell and Russell, J. Virol. (2000) 74:4612-4620; McCarty et al., Gene Ther. (2001) 8:1248-1254; each of which are hereby incorporated herein by reference in its entirety). Such preparations thus have largely varying efficiencies and require labor-intensive purification and enrichment steps.
Each of the following is hereby incorporated herein in its entirety by reference: U.S. Published Application Nos. 20020006664, 20030153519, and 20030139363; U.S. Pat. Nos. 6,547,099; 6,506,559; and 4,766,072; PCT Application Nos. WO 01/92551, WO 01/68836, and WO 03/010180. Recently, U.S. Published Application No. 20040029106 (“Samulsky”) teaches a modification termed double-stranded (ds) or self-complementary (sc) AAV, where one of the two AAV-2. ITRs carries a specific deletion of 6 nt, the so-called terminal resolution site (trs). The purpose of this deletion is as follows: during replication of a conventional ssAAV vector genome, a ds intermediate is formed and then subsequently resolved via nicking at the trs site. In the trs-deleted dsAAV version, this nicking reaction is ablated. As a result, the replication of these vectors becomes arrested at the ds intermediate step. This intermediate, containing two inverted copies of the recombinant gene expression cassette separated by the mutated ITR, then becomes encapsidated. In transduced cells, the two inverted copies of an expression cassette (each copy up to 2.2 kb in size) rapidly re-fold and anneal with each other, resulting in an immediate and strong onset of gene expression from dsAAV vectors. In contrast, standard ssAAV transduce substantially more slowly and less efficiently, because two individual complementary DNA strands have to “find” each other in the cell, which is a rate-limiting step.
Despite the increase in transduction efficacy, these dsAAV vectors share a substantial problem with ssAAV vectors—genetic instability during propagation, as observed in E. coli, as well as during in vitro manipulation of the vector plasmids. This is because both ss and dsAAV vectors carry identical (or nearly identical, when considering the 6 bp deletion in one ITR in dsAAV) ITRs, making them extremely prone to homologous recombination. A frequent result (˜50%) is either deletion of large parts (>20 bp) of one ITR, or gene conversion between the two ITRs. The latter is a particular problem with dsAAV, as it results in repair of the 6 bp deletion and consequently loss of the desired ds genotype.
Another related adverse consequence is a drop in vector particle titers. In fact, typical vector particle yields obtained with conventional dsAAV vectors are about 5-10 fold lower than what is possible with ssAAV vectors. Moreover, such standard dsAAV preparations usually contain a mixture of actual dsAAV genomes, together with half-sized monomers which result from ITR repair and subsequent nicking. And, it is very difficult to purify the wanted dsAAV vector particles from the contaminating monomers.
Moreover, the genetic instability of ss or dsAAV vectors with two (nearly) identical ITRs increases inversely with the insert size, making it impossible to clone inserts smaller than ˜2.5 kb into a conventional ssAAV vector, or smaller than ˜1 kb into dsAAV. Such minimal inserts are highly desirable for certain human gene therapy applications, where the recombinant gene expression cassette is small, such as for an interfering RNA (RNAi; typically <0.6 kb). In these cases, conventional vectors require the addition of stuffer DNA sequences, to increase the insert size to >1 (dsAAV) or 2.5 (ssAAV) kb. The presence of additional stuffer DNA is highly unwanted, as these sequences could cause serious adverse events in the patient, including an immune reaction.
Accordingly, one of skill will appreciate a nucleic acid construct that overcomes at least these limitations of both conventional ss or dsAAV vectors. The constructs taught herein overcome the problems of genetic instability, the resulting low titers, vector impurities and the need for stuffer DNA, thus providing a novel and valuable contribution to the art.